PROJECT SUMMARY Little is known about how bacteria prevalent in the digestive tract, most of which are anaerobes, take up and process the host?s dietary iron. How this activity influences the efficiency with which iron is absorbed by the host is also unclear. Closing this gap in the knowledge is of both fundamental and biomedical interest. Iron- deficiency and associated anemia are the most prevalent nutritional disorders worldwide, shared by nearly a third of the human population. At the same time, unmetabolized heme iron from red meat diets that remains in the colon has been associated with the development of diseases ranging from inflammation to colon cancer, with microbial activity postulated to play a key role. The long-term goal of this work is to understand how commensal bacteria commonly found in the healthy mammalian gut metabolize iron under the low/no O2 conditions that are prevalent in this ecosystem. The proposed work focuses our group?s knowledge and infrastructure ? accrued over 15 years of studying aerobic heme/iron biochemistry at the level of the catalyst, cell, and ecosystem ? on this ambitious long-term goal, which we have divided into two overlapping parts. First, we will examine how common gut microbes, most of which are anaerobic, heme auxotrophic bacteria (HAB), metabolize heme. We are focusing on three experimentally tractable HAB which are abundant in humans and which either require heme for respiration (Bacteroides thetaiotaomicron), are capable of but not dependent on heme-mediated respiration (Lactobacillus rhamnosus), or are obligately fermentative but still have limited uses for heme (Clostridium scindens). We will examine genes (via the generation of knock-outs) and gene products that are predicted to play important roles in heme metabolism in these species, but which belong to metabolic pathways that are typically incomplete. At the same time, we will employ discovery-based approaches to identify members of the heme-proteome, using chemically defined growth media, stable-isotope-labeled heme, and spectroscopic analyses with which we have a depth of expertise. Second, we will define how gut bacterial species work together and with the animal host to metabolize heme iron. As part of our experimental approach, we will use knock-out strains and isotopically labeled heme to examine heme metabolism by co-cultures, using subsets of the three HAB above and a common enteric heme heterotroph (Escherichia coli). Cocultures will be studied both in the flask and in mice with defined (gnotobiotic) microbiomes, in collaboration with Prof. Seth Walk (MSU). Understanding anaerobic heme metabolism by commensal bacteria serves the long-term biomedical goal of manipulating the microbiome to facilitate host metabolism of iron, thereby remediating diseases associated with iron deficiency (anemia) or excess (infection, colitis, inflammation, colon cancer).